H3K9 Demethylases JMJD1A and JMJD1B Control Prospermatogonia to Spermatogonia Transition in Mouse Germline

Histone H3 lysine 9 (H3K9) methylation is dynamically regulated by methyltransferases and demethylases. In spermatogenesis, prospermatogonia differentiate into differentiating or undifferentiated spermatogonia after birth. However, the epigenetic regulation of prospermatogonia to spermatogonia transition is largely unknown. We found that perinatal prospermatogonia have extremely low levels of di-methylated H3K9 (H3K9me2) and that H3K9 demethylases, JMJD1A and JMJD1B, catalyze H3K9me2 demethylation in perinatal prospermatogonia. Depletion of JMJD1A and JMJD1B in the embryonic germline resulted in complete loss of male germ cells after puberty, indicating that H3K9me2 demethylation is essential for male germline maintenance. JMJD1A/JMJD1B-depleted germ cells were unable to differentiate into functional spermatogonia. JMJD1 isozymes contributed to activation of several spermatogonial stem cell maintenance genes through H3K9 demethylation during the prospermatogonia to spermatogonia transition, which we propose is key for spermatogonia development. In summary, JMJD1A/JMJD1B-mediated H3K9me2 demethylation promotes prospermatogonia to differentiate into functional spermatogonia by establishing proper gene expression profiles

The effects of short-term hypoxia on human mesenchymal stem cell proliferation, viability and p16(INK4A) mRNA expression: Investigation using a simple hypoxic culture system with a deoxidizing agent

A hypoxic environment is thought to be important for the maintenance of stemness and suppressing cell senescence, in stem cells. Therefore, a hypoxic condition is induced during cell expansion and/or induction of intended differentiation. However, the induction of these conditions requires a specially equipped hypoxia chamber and expensive gas mixtures, which are expensive and space-consuming. Owing to these restrictions, appropriate hypoxic conditions cannot be provided during cell transportation, which is increasingly required for regenerative medicine. Hence, a simple and economical culture system is required. The purpose of this study was to investigate the effects of short-term hypoxic conditions on human mesenchymal stem cell (MSC) proliferation, viability, and senescence, utilizing the CulturePal system (CulturePal-Zero and CulturePal-Five), a novel and simple hypoxic culture system with a built-in deoxidizing agent. The O₂ concentration in the CulturePal-Zero was observed to reduce to <0.1% within 1 h, and to 5% within 24 h in the CulturePal-Five system. Cell proliferation under these hypoxic conditions showed a sharp increase at 5% O₂ concentration, and no noticeable cell death was observed even at severe hypoxic conditions (<0.1% O₂) up to 72h. The p16INK4A (cell senescence marker) mRNA expression was retained under hypoxic conditions up to 72h, but it was up-regulated under normoxic conditions. Interestingly, the p16INK4A expression altered proportionately to the O₂ concentration. These results indicated that the short-term hypoxic condition, at an approximate O₂ concentration of 5%, would be suitable for promoting cell proliferation and repressing cell senescence, without aggravating the MSC viability. Therefore, the CulturePal systems may be suitable for providing an appropriate hypoxic condition in stem cell research and transportation

Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture

ヒトのiPS細胞から腱の細胞を作製する --アキレス腱断裂のラットに移植し、機能回復を確認--. 京都大学プレスリリース. 2021-08-31.Repairing tendon injuries with stem cells. 京都大学プレスリリース. 2021-08-31.Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury

Acoustic stiffness and change in plug cartilage over time after autologous osteochondral grafting: correlation between ultrasound signal intensity and histological score in a rabbit model

We investigated quantitative changes over time in ultrasound signal intensity (an index of stiffness), signal duration (an index of surface irregularity), and interval between signals (an index of thickness) of plug cartilage in an animal model of autologous osteochondral grafting. A full-thickness osteochondral plug was surgically removed and replaced in male Japanese white rabbits (n = 22). Specimens obtained at day 0 and weeks 2, 4, 8, 12 and 24 postoperatively were assessed using an ultrasound system and by macroscopic and histological evaluation (modified Mankin's score). Histology revealed that the plug sank until 2 weeks postoperatively, and that newly formed cartilage-like tissue covered the plug, but at 24 weeks the tissue detached. The plug itself survived well throughout the period of observation. Although the signal intensity at the plug site was same as that in the sham operated contralateral knee at day 0, from 2 to 24 weeks postoperatively it was less than that in the sham knee. At 8 weeks, this difference was significant (P < 0.05). Modified Mankin's score revealed early degenerative changes at the site, but macroscopic examination did not. Signal intensity correlated significantly with score (both at day 0 and at the five postoperative time points [P < 0.05, r = -0.91] and as a whole [P < 0.05, r = -0.36]). Signal intensity also significantly correlated with the individual subscores for 'cartilage structure' (P < 0.05, r = -0.32) and 'cartilage cells' (P < 0.05, r = -0.30) from the modified Mankin's score, but not significantly with subscores for 'staining' and 'tidemark'. Signal duration correlated significantly with total score (as a whole [P < 0.05, r = 0.34]), but not significantly with the score for cartilage structure (P = 0.0557, r = 0.29). The interval between signals reflected well the actual thickness of the plug site. The significant relationships between ultrasound signal intensity and scores suggest that early degenerative changes in plug cartilage and cartilage-like tissue, especially in the superficial layer, are detectable by high-frequency ultrasound assessment

Tuning of Sry expression by H3K9 methylation and demethylation

Histone H3 lysine 9 (H3K9) methylation is a hallmark of heterochromatin. H3K9 demethylation is crucial in mouse sex determination; The H3K9 demethylase Jmjd1a deficiency leads to increased H3K9 methylation at the Sry locus in embryonic gonads, thereby compromising Sry expression and causing male-to-female sex reversal. We hypothesized that the H3K9 methylation level at the Sry locus is finely tuned by the balance in activities between the H3K9 demethylase Jmjd1a and an unidentified H3K9 methyltransferase to ensure correct Sry expression. Here we identified the GLP/G9a H3K9 methyltransferase complex as the enzyme catalyzing H3K9 methylation at the Sry locus. Based on this finding, we tried to rescue the sex-reversal phenotype of Jmjd1a-deficient mice by modulating GLP/G9a complex activity. A heterozygous GLP mutation rescued the sex-reversal phenotype of Jmjd1a-deficient mice by restoring Sry expression. The administration of a chemical inhibitor of GLP/G9a enzyme into Jmjd1a-deficient embryos also successfully rescued sex reversal. Our study not only reveals the molecular mechanism underlying the tuning of Sry expression but also provides proof on the principle of therapeutic strategies based on the pharmacological modulation of epigenetic balance

Genomic surveillance of Neisseria gonorrhoeae to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages

The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial-susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85 % of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99 % sensitivity and 100 % specificity. Approximately 10 % showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide

Combined Loss of JMJD1A and JMJD1B Reveals Critical Roles for H3K9 Demethylation in the Maintenance of Embryonic Stem Cells and Early Embryogenesis

Histone H3 lysine 9 (H3K9) methylation is unevenly distributed in mammalian chromosomes. However, the molecular mechanism controlling the uneven distribution and its biological significance remain to be elucidated. Here, we show that JMJD1A and JMJD1B preferentially target H3K9 demethylation of gene-dense regions of chromosomes, thereby establishing an H3K9 hypomethylation state in euchromatin. JMJD1A/JMJD1B-deficient embryos died soon after implantation accompanying epiblast cell death. Furthermore, combined loss of JMJD1A and JMJD1B caused perturbed expression of metabolic genes and rapid cell death in embryonic stem cells (ESCs). These results indicate that JMJD1A/JMJD1B-meditated H3K9 demethylation has critical roles for early embryogenesis and ESC maintenance. Finally, genetic rescue experiments clarified that H3K9 overmethylation by G9A was the cause of the cell death and perturbed gene expression of JMJD1A/JMJD1B-depleted ESCs. We summarized that JMJD1A and JMJD1B, in combination, ensure early embryogenesis and ESC viability by establishing the correct H3K9 methylated epigenome

FGF2 Has Distinct Molecular Functions from GDNF in the Mouse Germline Niche

Both glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells, whereas retinoic acid (RA) induces spermatogonial differentiation. In this study, we investigated the functional differences between FGF2 and GDNF in the germline niche by providing these factors using a drug delivery system in vivo. Although both factors expanded the GFRA1+ subset of undifferentiated spermatogonia, the FGF2-expanded subset expressed RARG, which is indispensable for proper differentiation, 1.9-fold more frequently than the GDNF-expanded subset, demonstrating that FGF2 expands a differentiation-prone subset in the testis. Moreover, FGF2 acted on the germline niche to suppress RA metabolism and GDNF production, suggesting that FGF2 modifies germline niche functions to be more appropriate for spermatogonial differentiation. These results suggest that FGF2 contributes to induction of differentiation rather than maintenance of undifferentiated spermatogonia, indicating reconsideration of the role of FGF2 in the germline niche

Protocol for a multicentre, prospective, cohort study to investigate patient satisfaction and quality of life after immediate breast reconstruction in Japan: the SAQLA study

Introduction The aim of breast reconstruction (BR) is to improve patients' health-related quality of life (HRQOL). Therefore, measuring patient-reported outcomes (PROs) would clarify the value and impact of BR on a patient's life and thus would provide evidence-based information to help decision-making. The Satisfaction and Quality of Life After Immediate Breast Reconstruction study aimed to investigate satisfaction and HRQOL in Japanese patients with breast cancer who undergo immediate breast reconstruction (IBR). Methods and analysis This ongoing prospective, observational multicentre study will assess 406 patients who had unilateral breast cancer and underwent mastectomy and IBR, and were recruited from April 2018 to July 2019. All participants were recruited from seven hospitals: Okayama University Hospital, Iwate Medical University Hospital, The Cancer Institute Hospital of Japanese Foundation for Cancer Research, Showa University Hospital, University of Tsukuba Hospital, Osaka University Hospital and Yokohama City University Medical Center. The patients will be followed up for 36 months postoperatively. The primary endpoint of this study will be the time-dependent changes in BREAST-Q satisfaction with breast subscale scores for 12 months after reconstructive surgery, which will be collected via an electronic PRO system. Ethics and dissemination This study will be performed in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects published by Japan's Ministry of Education, Science and Technology and the Ministry of Health, Labour and Welfare, the modified Act on the Protection of Personal Information and the Declaration of Helsinki. This study protocol was approved by the institutional ethics committee at the Okayama University Graduate School of Medicine, Dentistry, on 2 February 2018 (1801-039) and all other participating sites. The findings of this trial will be submitted to an international peer-reviewed journal